Biomedical subjects

J Anné

Publications and source records attributed to J Anné.

At least 19 recordsLinked to original sources

PubMed2006

Repeated cycles of Clostridium-directed enzyme prodrug therapy result in sustained antitumour effects in vivo.

The unique properties of the tumour microenvironment can be exploited by using recombinant anaerobic clostridial spores as highly selective gene delivery vectors. Although several recombinant Clostridium species have been generated during the past decade, their efficacy has been limited. Our goal was to substantially improve the prospects of clostridia as a gene delivery vector. Therefore, we have assessed a series of nitroreductase (NTR) enzymes for their capacity to convert the innocuous CB1954 prodrug to its toxic derivative. Among the enzymes tested, one showed superior prodrug turnover characteristics. In addition, we established an efficient gene transfer procedure, based on conjugation, which allows for the first time genetic engineering of Clostridium strains with superior tumour colonisation properties with high success rates. This conjugation procedure was subsequently used to create a recombinant C. sporogenes overexpressing the isolated NTR enzyme. Finally, analogous to a clinical setting situation, we have tested the effect of multiple consecutive treatment cycles, with antibiotic bacterial clearance between cycles. Importantly, this regimen demonstrated that intravenously administered spores of NTR-recombinant C. sporogenes produced significant antitumour efficacy when combined with prodrug administration.

Animals↗

PubMed2006

The use of clostridial spores for cancer treatment.

Hypoxic/necrotic regions, absent in normal tissues, can be exploited to target tumours in cancer therapy using nonpathogenic strains of the bacterial genus Clostridium. Following administration of Clostridium spores to tumour-bearing organisms, these spores can only germinate within the hypoxic/necrotic regions of solid tumours, proving their exquisite selectivity. Low oxygen tension is a common feature of solid tumours, which may arise from the unique physiological environment, generated to a large extent by the abnormal tumour vasculature, and provides as such a niche for anaerobic bacteria. Some clostridia tested clearly showed innate oncolytic activity, but they could not completely eradicate the tumour. Recombinant clostridia producing prodrug-converting enzymes or cytokines resulted in the production of such proteins solely within the tumour, and where applicable, could convert the prodrug in a toxic compound. Moreover, in some cases, tumour eradication or tumour control could be observed. This review brings an overview of the relative successes and failures of the Clostridium-directed tumour therapy with both wild-type strains and strains producing proteins useful in antitumour therapy.

Clostridium↗

PubMed2005

Complete genomic nucleotide sequence and analysis of the temperate bacteriophage VWB.

The entire double-stranded DNA genome of the Streptomyces venezuelae bacteriophage VWB was sequenced and analyzed. Its size is 49,220 bp with an overall molar G + C content of 71.2 mol%. Sixty-one potential open reading frames were identified and annotated using several complementary bioinformatics tools. Clusters of functionally related putative genes were defined, supporting a refined version of the modular theory of phage evolution.

Amino Acid Sequence↗

PubMed2004

Isolation of high quality RNA from Streptomyces.

Several molecular techniques require high quality RNA, completely free of DNA. Standard methods to isolate total RNA from Streptomyces spp. are based on the application of a 'Modified Kirby Mix' [Practical Streptomyces Genetics. The John Innes Foundation, Norwich, pp. 613]. Here we present an alternative procedure using Triton X-100 and EDTA for the isolation of total RNA from Streptomyces.

Edetic Acid↗

PubMed2003

Non-invasive 19F MR spectroscopy of 5-fluorocytosine to 5-fluorouracil conversion by recombinant Salmonella in tumours.

The aim of this study was to evaluate the applicability of fluorine-19 magnetic resonance spectroscopy ((19)F MRS) for monitoring in vivo the conversion of 5-fluorocytosine (5-FC) to 5-fluorouracil (5-FU) after using an attenuated Salmonella Typhimurium strain recombinant to provide cytosine deaminase (TAPET-CD). The (19)F MRS measurements were done on mice bearing the human colon tumour xenograft (HCT116). The intratumoural conversion is greater when TAPET-CD/5-FC is delivered intratumourally (i.tu.) than when TAPET-CD is delivered intravenously (i.v.) and 5-FC intraperitoneally (i.p.). Repeat measurements of the same tumour also yielded important information on the tumour colonization by TAPET-CD through the correlated 5-FC to 5-FU conversion efficacy. The in vivo MRS spectra were confirmed by in vitro (19)F MRS of perchloric acid extracts of the tumour tissue. No 5-FU metabolites were detectable in vivo in the tumours. However, the in vitro measurements revealed, besides 5-FC and 5-FU, the presence of small amounts of catabolites. Finally, spectra obtained in vitro from liver extracts of tumour-bearing mice treated i.tu. with TAPET-CD/5-FC showed no 5-FU and only little amounts of catabolites. Our data illustrate most importantly the potential of (19)F MRS to monitor biologically-based treatments involving cytosine deaminase.

Animals↗

PubMed2003

Improved PCR-based method for the direct screening of Streptomyces transformants.

Streptomycetes are attractive microorganisms because of their high secretion capacity and for the production of secondary metabolites. We report the improvement of a PCR-based method for screening of solid media-grown Streptomyces transformants. By resuspending mycelium into 2.5-10% dimethyl sulfoxide (DMSO), extensive manipulation prior to PCR could be avoided, thus significantly reducing screening time. Results proved to be both reliable and specific.

Dimethyl Sulfoxide↗

PubMed2003

Real-time quantitative RT-PCR and detection of tumour cell dissemination in breast cancer patients: plasmid versus cell line dilutions.

BACKGROUND: We previously developed a real-time quantitative RT-PCR technique to detect breast carcinoma cells in peripheral blood (PB). The aim of the current study was to improve cytokeratin 19 (CK19) quantification using plasmid dilutions of cloned PCR fragments to obtain a more reliable and reproducible quantification of CK19 transcripts. MATERIALS AND METHODS: PB samples of 14 stage IV breast cancer patients and 23 healthy controls were examined with RT-PCR using plasmid quantification. RESULTS: Median CK19+ copy numbers of one and 11 were detected in the control group and stage IV breast cancer patients, respectively (Mann-Whitney, P </=0.0001). When comparing the results obtained using cell line dilutions with those obtained using plasmid dilutions, a good correlation was observed (r(2) = 0.98). CONCLUSIONS: Plasmid dilutions are more reliable than cell line dilutions for quantification of gene expression, and more objective criteria for positivity could be defined based on the characteristics of the standard curve (slope and intercept). A more universally accepted agreement on the definition of the cut-off value for positivity is needed.

Breast Neoplasms↗

PubMed2003

Tumor-specific gene delivery using genetically engineered bacteria.

The loco-regional control of cancer remains a major contributor to the treatment outcome for many cancer patients prescribed conventional radiotherapy or chemotherapy. Failure of treatment coupled with the realisation that cancer is essentially a genetic disease has led to development of many clinical protocols based on gene therapy. In this review, we will describe an alternative gene delivery system based on the use of non-pathogenic bacteria. Tumor regressions have been reported long ago in patients with bacterially infected tumors, suggesting that bacteria could target tumors and have local anti-tumor effects. The basis of this phenomenon is attributable to the unique properties of the tumor micro-environment. The presence of hypoxic and/or necrotic areas provides a haven for a number of anaerobic bacteria and over the past 60 years, several strains of anaerobic bacteria have been shown to localise within and cause cell lysis of experimental animal tumors. One of the most important strains in that context is Clostridium. Other bacteria have also been implicated in experimental anti-cancer settings. Of these, attenuated Salmonella strains capable of both selective amplification within tumors and expression of effector genes encoding therapeutic proteins are probably the most promising. We will discuss the potential advantages and the pitfalls of this alternative delivery approach. We will emphasize the importance of hypoxia in solid tumors and discuss the potential of radiation-inducible promoters and combined treatment modalities, involving vascular targeting and radiotherapy. We believe that this approach will act in a complementary way to current radiotherapy and chemotherapy treatments of solid tumors.

Animals↗

PubMed2002

Clostridium spores for tumor-specific drug delivery.

Insufficient blood supply of rapidly growing tumors leads to the presence of hypoxia, a well-known feature in solid tumors. Hypoxia is known to decrease the efficiency of currently used anti-cancer modalities like surgery, chemotherapy and radiotherapy. Therefore, hypoxia seems to be a major limitation in current anti-cancer therapy. The use of non-pathogenic clostridia to deliver toxic agents to the tumor cells takes advantage of this unique physiology. These strictly anaerobic, Gram-positive, spore-forming bacteria give, after systemic administration, a selective colonization of hypoxic/necrotic areas within the tumor. Moreover, they can be genetically modified to secrete therapeutic proteins like cytosine deaminase or tumor necrosis factor-alpha. The specificity of this protein delivery system can be further increased when expression is controlled by the use of a radio-inducible promoter, leading to increased spatial and temporal regulation of protein expression. This approach of bacterial vector systems to target protein expression to the tumor can be considered very safe since bacteria can be eliminated at any moment by the addition of proper antibiotics. The Clostridium-based delivery system thus presents an alternative therapeutic modality to deliver anti-tumor agents specifically to the tumor site. This high selectivity offers a major advantage in comparison with the classical gene therapy systems.

Animals↗

PubMed2001

Protein secretion biotechnology using Streptomyces lividans: large-scale production of functional trimeric tumor necrosis factor alpha.

We evaluated the feasibility of large-scale production of biopharmaceuticals expressed as heterologous polypeptides from the Gram-positive bacterium Streptomyces lividans. As a model protein we used murine tumor necrosis factor alpha (mTNFalpha). mTNFalpha fused C-terminally to the secretory signal peptide of the subtilisin-inhibitor protein from Streptomyces venezuelae. Under appropriate fermentation conditions, significant amounts of mature mTNFalpha (80-120 mg/L) can be recovered from spent growth media. Efficient downstream processing allowing rapid purification of mTNFalpha from culture supernatants was developed. Importantly, the protein is recovered from the spent growth medium in its native trimeric state as judged by biophysical analysis. Further, mTNFalpha secreted by S. lividans is significantly more active in an in vitro apoptosis tissue culture assay than a corresponding polypeptide produced in Escherichia coli. This pilot study provides the first validation of S. lividans protein secretion as an alternative bioprocess for large-scale production of oligomeric proteins of potential therapeutic value.

Bioreactors↗

PubMed2001

In vivo antitumor effect of vascular targeting combined with either ionizing radiation or anti-angiogenesis treatment.

PURPOSE: Interference with the tumor blood vessels through anti-angiogenesis or vascular targeting can indirectly suppress tumor growth. Vascular targeting of solid tumors, using tubulin-compromising agents, seems a promising and selective novel treatment. We aimed to evaluate the potential (hypothesis-based) benefit from combinations of vascular targeting using combretastatin A-4 phosphate (combreAp) with either ionizing radiation or anti-angiogenesis. METHODS AND MATERIALS: Rhabdomyosarcoma tumor pieces were inplanted subcutaneously (s.c.) in the lower flank region of syngeneic adult WAG/Rij rats. Tumors were grown until different sizes and stratified for the various treatment groups: small (1-3 cm3), medium (3.1-7 cm3), and large (7.1-14 cm3). CombreAp was injected i.p.; injections of TNP-470 were s.c. in the neck area. Localized single-dose (8 Gy) irradiations of tumors were done under Nembutal anesthesia, always 1 day before a single combreAp (25 mg/kg) injection. The TNP-470 treatment (3 times 30 mg/kg in 1 week) started 1 day after a double (8 days interval between both) combreAp administration. Tumor responses were evaluated by the growth delay assay, and statistical significance of tumor growth change was computed. RESULTS: Large tumors responded better to combreAp treatment given alone than did the smaller ones, confirming our previous data with this tumor model. Combining irradiation with combreAp also resulted in a tumor size-dependent growth delay. With small and medium tumor volumes, a similar response was measured after the combination treatment when compared with irradiation only. Large tumors, however, showed a strong (at least additive) increase of the growth delay with the combined therapy; the difference in tumor growth between the two treatment groups was very significant (p < 0.0001). m When TNP-470 was combined with combreAp, no significant lengthening of the growth delay, irrespective of the tumor size, was present with the applied schedule. CONCLUSION: The current data show a significant advantage in the combination of combreAp with irradiation in rhabdomyosarcomas having a large size (7-14 cm3) at treatment. Such a benefit in tumor response was not observed with the smaller tumors, seemingly because irradiation as such was very effective. No significant gain in growth delay for any tumor size was observed when TNP-470, showing efficacy on its own specifically with tumors measuring <7 cm3, was added to the combreAp treatment. This presumably reflects only little angiogenesis during the first week of rhabdomyosarcoma regrowth after the combreAp treatment.

Angiogenesis Inhibitors↗

PubMed2001

Functional analysis of the Streptomyces lividans type I signal peptidases.

Type I signal peptidases are responsible for the proteolytic cleavage of the signal peptide of secreted proteins. In the gram-positive bacterium Streptomyces lividans, four adjacent genes (sipW, sipX, sipY and sipZ) were isolated encoding putative type I signal peptidases. In this work, the different sip genes were cloned and expressed. Subsequently, the Sip proteins were purified to raise antibodies. Although the four Sip proteins share a low degree of sequence similarity and differ significantly in size and pI, anti-Sip antibodies cross-reacted intensively. Functional signal peptidase processing activity for each of these Sip proteins was shown both in vitro and in vivo. The different Sip proteins did not exhibit the same cleavage efficiency on the Bacillus subtilis pre-chitosanase.

Antibodies, Bacterial↗

PubMed2001

Efficient isolation of total RNA from Clostridium without DNA contamination.

Several molecular techniques require high-quality RNA, free from DNA. Various methods have been described to obtain RNA to be used in expression studies or as starting material in differential display-reverse transcriptase (dd-RT-PCR), for which high-quality RNA free from DNA is an essential requirement. In this report, we compare three different methods to isolate RNA from Gram-positive bacteria: (1) An acid-phenol extraction protocol. (2) The "RNeasy mini kit" from QIAGEN (Valencia, CA, USA). (3) The "SV Total RNA Isolation System" from Promega (Madison, WI, USA).The QIAGEN-kit delivers the highest amount of RNA with the highest purity. Slot blot analysis and dd-RT-PCR confirm the absence of DNA contamination and Northern blot analysis and dd-RT-PCR show high quality of the extracted RNA. This RNA extraction method thus addresses current problems by permitting rapid and safe isolation with high yields of intact RNA for subsequent analysis.

Blotting, Northern↗

PubMed2001

Specific targeting of cytosine deaminase to solid tumors by engineered Clostridium acetobutylicum.

The presence of severe hypoxia and necrosis in solid tumors offers the potential to apply an anaerobic bacterial enzyme/prodrug approach in cancer treatment. In this context the apathogenic C. acetobutylicum was genetically engineered to express and secrete E. coli cytosine deaminase (CDase). Considerable levels of functional cytosine deaminase were detected in lysates and supernatants of recombinant C acetobutylicum cultures. After administration of the recombinant Clostridium to rhabdomyosarcoma bearing rats used as a model, cytosine deaminase could be detected at the tumor site. Moreover, following administration of the vascular targeting agent combretastatin A-4 phosphate significantly increased levels of cytosine deaminase were detected at the tumor site as a consequence of enlarged tumor necrosis and subsequently improved growth of C. acetobutylicum. The results provide evidence for the potential application of Clostrisdium-based therapeutic protein transfer to tumors in anticancer therapy.

Animals↗

PubMed2001

Radio-responsive recA promoter significantly increases TNFalpha production in recombinant clostridia after 2 Gy irradiation.

One of the major problems with gene therapy today is the lack of tumour specificity. The use of anaerobic apathogenic clostridia as a gene transfer system can target anoxic areas within the tumour. These bacteria can be genetically modified to express therapeutic proteins such as TNFalpha locally in the tumour. As shown in our results, ionising irradiation can be used in clostridia to activate genes encoding cytotoxic agents under control of a radiation-inducible promoter. A 44% significant increase (P < 0.05) in TNFalpha secretion was seen 3.5 h after a single dose of 2 Gy. A second dose of 2 Gy was also capable of repeating gene activation and gave a significant increase of TNFalpha production of 42% (P < 0.05). These results provide evidence that spatial and temporal control of gene expression can be achieved using a radio-inducible promoter. Repetitive gene activation was feasible with a second dose of 2 Gy, indicating that fractionated radiotherapy could lead to repeated gene induction resulting in prolonged and enhanced protein expression. Gene targeting by ionising radiation could thus provide a new means of increasing the therapeutic ratio in cancer treatment.

Clostridium↗

PubMed2001

Effect of TNP-470 (AGM-1470) on the growth of rat rhabdomyosarcoma tumors of different sizes.

Potential anticancer therapy with the fumagillin analog TNP-470 was investigated in the present project using subcutaneously growing rhabdomyosarcomas in rats. Specifically, influences of different tumor sizes at the start of treatment as well as dose/schedules were evaluated with this angiogenesis inhibitor. The results show a significant (p = < or = 0.01) reduction of the growth rate, even for relatively large-sized (> 7 cm3) tumors, when 50 mg/kg TNP-470 was used every other day for up to 3 or 5 injections. With 30 mg/kg TNP-470 injections, effects were seen only with tumors measuring < 7 cm3. The histologic examinations demonstrate an increase in necrosis, both in the center and in the peripheral part of TNP-470-treated tumors. Overall, both tumor volume and drug dose determine treatment outcome with the rat rhabdomyosarcoma. The results suggest that angiogenesis inhibitors could represent a valid component in the treatment of progressive tumor growth, also of large tumors as often encountered in clinics. The antivasculature therapy might also improve hypoxia/necrosis-related therapeutic approaches.

Angiogenesis Inhibitors↗

PubMed2001

Improvement of Clostridium tumour targeting vectors evaluated in rat rhabdomyosarcomas.

Previous studies have demonstrated the feasibility of using apathogenic clostridia as a promising strategy for hypoxia-specific tumour targeting. The present study shows that the use of the vascular targeting compound combretastatin A-4 phosphate could significantly (P<0.001) increase the number of Clostridium vegetative cells in rat rhabdomyosarcomas with sizes between 0.2 cm(2) and 3 cm(2). Furthermore, this study showed that administration of metronidazole for a 9-day period was sufficient to eliminate systemically administered Clostridium from the tumour. Moreover, previous Clostridium spore administration did not effect tumour colonisation, regardless of the immune response status of the host.

Animals↗

PubMed2001

Insertion or deletion of the Cheo box modifies radiation inducibility of Clostridium promoters.

Radiation-inducible promoters are being used in many viral vector systems to obtain spatial and temporal control of gene expression. It was previously proven that radiation-induced gene expression can also be obtained in a bacterial vector system using anaerobic apathogenic clostridia. The effect of radiation inducibility was detected using mouse tumor necrosis factor alpha (mTNF-alpha) as a model protein under regulation of the radiation-inducible recA promoter. In this report, experiments are described in which this recA promoter was modified in order to increase radiation responsiveness. Incorporation of an extra Cheo box in the recA promoter region resulted in an increase in mTNF-alpha secretion from 44% for the wild-type promoter to 412% for the promoter with an extra Cheo box after a single irradiation dose of 2 Gy. Deletion of the Cheo box in the promoter region eliminated radiation inducibility. These results prove that the Cheo box in the recA promoter is indeed the radiation-responsive element. We also tested whether we could induce the constitutive endo-beta-1,4-glucanase promoter (eglA) via ionizing irradiation by introducing a Cheo box in the promoter region. While the use of the constitutive promoter did not lead to an increase in mTNF-alpha secretion after irradiation, the introduction of a Cheo box resulted in a 242% increase in mTNF-alpha secretion. Reverse transcriptase PCR of RNA samples isolated from irradiated and nonirradiated bacterial cultures demonstrated that the increase in secretion was the result of enhanced transcription of the mTNF-alpha gene.

Animals↗